The key feature for Francisella tularensis pathogenesis is its ability to survive inside the macrophage. Studies with other bacterial intracellular parasites have demonstrated that such a feat is usually accomplished by the mode of entry into the host cell and by bacterial virulence factors that specifically usurp host cell signaling pathways to position the bacterium-containing vacuole within protected coordinates unique to each bacterium within the host cell. Very few such factors have so far been defined in F. tularensis. This project proposes that F. tularensis steers its evolving phagosome to a defined and unique protected position within the macrophage through the elaboration of specific virulence factors that determine its mode of entry and target host cell signaling pathways. We will test this hypothesis by characterizing the F. tularensis-containing vacuole by biochemical and cell biology analyses to understand the trafficking and maturation of the phagosome. The F. tularensis Schu4 strain, which is virulent for humans, will be used in all studies. The factors that contribute to F. tularensis intracellular survival will be characterized by genetic and biochemical techniques. The entry pathway of the bacterium will be investigated by electron microscopy, manipulation of the entry pathway through selective host cell receptor-blockade, and investigation of the cell signaling pathways activated via entry of F. tularensis. An understanding of the pathway of F. tularensis from the initial contact with the macrophage surface to its subcellular localization of protected replication within the macrophage and knowledge of the bacterial virulence factors involved are an important prerequisite for developing vaccines and novel antibacterial components. The investigations proposed here are within the overall goal of our laboratory to characterize the vesicle trafficking pathway within macrophages as it relates to invading intracellular microbes.